The recent threat of bioterrorism has intensified the need for new classes of antibiotics. Among biodefense category A bacterial pathogens, many species are currently susceptible to existing antibiotics. However, the development of bioweapon strains of Bacillus anthracis and Yersinia pestis that are resistant to multiple antibiotics is a distinct possibility. In addition to agents present on the biodefense pathogen list, other forms of bacterial infections continue to be a common cause of morbidity and mortality. To begin addressing these unmet needs, we are proposing a focused study targeting bacterial mechanosensitive (MS) channels. MS channels enable bacteria to rapidly adapt to osmotic changes in their environment. Although non-essential, gain-of-function (GOF) mutations that constitutively activate MS channels are bactericidal. Over the initial phase of this project, we will demonstrate the "drugability" of the E. coil MS channel using our proprietary Mpex minicell technology. The Mpex minicell will enable high-throughput, small molecule and natural product screening to identify compound activators that mimic the lethal GOF phenotype. In conjunction with these studies, we will validate the function of homologous, biodefense-related MS channels from B. anthracis and Y. pestis. Using E. coil and Mpex minicells, we will also characterize the GOF modulators identified against the E. coil MS target for activity against these pathogenic MS channeling proteins. Thus, Phase I stands as a proof-of-concept for the use of the MS target and Mpex minicells together to effectively identify novel antibacterial leads, and provides in parallel further functional target validation of MS homologs found in various biodefense category A pathogenic species. Following successful validation in Phase I, Phase II studies will include screening of these pathogenic MS homologs using Mpex minicells, prioritization of hits, and lead optimization to produce an IND-enabling candidate molecule.